miRNA介导TGF-beta1→VEGFA调控通路在猪卵泡闭锁发生过程中的作用

Studies on the mechanism of follicular atresia are important for improving the fertility of livestock and animal reproductive health. Our previous studies demonstrated that VEGFA were significantly differential expression between health and early follicular atresia follicles in pig, and the expression of TGF-beta1/Smad family members was regulated by miRNA molecular, which was closely related to granulosa cell apoptosis. In addition, there were reports in human TGF-beta1 could control the expression of VEGFA by beta-catenin and Smad proteins, significant interaction existed among beta-catenin, smads and TCF4. Furthermore, there were two TCF binding sites in the promoter region of VEGFA. Therefore, we hypothesized that: TGF-beta1/Smad/beta-catenin/TCF regulatory pathway mediated by miRNA plays a key role for VEGFA expression in granulosa cells, affects the follicular angiogenesis and ultimately determine the fate of individual follicle. Based on the above assumption, we intend to undertake the following tasks in this project: 1) by small RNA deep sequencing and bioinformatic analysis, we identify the key miRNAs targeting TGF-beta1→VEGFA regulatory pathway during porcine follicular atresia; 2) using the technology or method such as qRT-PCR, WB, fluorescent luciferase reporter vector, ChIP, CoIP, Immunohistochemistry, knockdown and overexpression and so on, we elucidate the effects of specific miRNA and the underlying mechanism involved in the above-described regulatory pathway in porcine granulosa cell; 3) by FACS, RNA interference and in vitro antral follicle culture, we reveal the exact role of the whole regulatory pathway for porcine granulosa cell apoptosis and follicular atresia. The project offers an useful complement for research on regulatory mechanism of follicular atresia, also provides the necessary theoretical basis for further manual intervention of follicular atresia process and fully exploiting mammal follicle reserves.

我们研究证实：VEGFA在健康和早期闭锁卵泡中显著差异表达；TGF-bata和Smad家族成员的表达受到miRNA调控,与颗粒细胞凋亡密切关联。在人类有报道TGF-beta1通过beta-catenin 和Smad蛋白调控VEGFA表达。我们推测：miRNA能介导TGF-beta1/Smad/beta-catenin/TCF通路对颗粒细胞VEGFA表达起调控作用，从而影响卵泡发育、闭锁进程。基于此，本项目拟开展工作：1）采用小RNA测序、生物信息分析，解析猪卵泡闭锁发生过程中靶向TGF-beta1→VEGFA通路关键miRNA；2）采用qRT-PCR、WB、荧光素酶报告载体、ChIP、CoIP、免疫组化及抑制、过表达等方法，明确关键miRNA在颗粒细胞参与上述调控通路的作用及机制；3）采用FACS、小RNA干涉及卵泡体外培养等技术验证调控通路在猪卵泡颗粒细胞凋亡及卵泡闭锁发生过程中的作用。

本项目针对“解析猪卵泡闭锁发生过程中靶向TGF-β→VEGFA通路的关键miRNA分子”和“关键miRNAs特异性参与TGF-β→VEGFA调控通路的作用及机制”的研究主题和目标，通过小RNA深度测序技术，鉴定了不同闭锁程度卵泡中所表达的miRNAs分子，探索猪卵巢卵泡中的miRNA与卵泡闭锁和颗粒细胞凋亡的关系。同时，结合不同闭锁程度猪卵泡中基因转录特性综合分析，发掘了涉及猪卵巢卵泡闭锁起始的潜在基因调控网络。我们筛选了靶向TGF-β→VEGFA通路关键信号分子的特异性miRNA，主要包括：miR-181b、miR-10a-5p、miR-425、miR-10b、miR-143、miR-361-5p。对特异性miRNA与通路靶基因的调控关系进行综合分析及验证,我们明确了上述特异性miRNA分子与SMAD4、SMAD7、TGFBR1和TGFBR2等TGF-β信号通路分子及相关基因CYP19A1、FSHR、INHA、CTGF以及VEGFA的靶向调控关系。同时，结合前述调控网络及TGF-β信号通路的关键节点，检测它们在卵巢卵泡血管生成、生长和闭锁中的作用和相互联系，解析了各靶点在猪卵泡闭锁过程中的表达及分布特征，阐明了其在调控猪卵泡颗粒细胞凋亡及卵泡闭锁过程中的重要生物学作用，鉴定出了关键miRNA介导TGFβ信号通路在猪卵泡颗粒细胞凋亡及卵泡闭锁过程中的作用途径及机制。